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The history, chemistry and modes of action of carmine and related dyes

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The history, chemistry and modes of action of carmine
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of carmine and related dyes', Biotechnic and Histochemistry, 82:4, 173 - 187
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 The history, chemistry and modes of action of
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carmine and related dyes

RW Dapson
Dapson & Dapson LLC, 6951 East AB Avenue, Richland, MI 49083

Submitted March 14, 2007; revised May 29, 2007; accepted May 30, 2007

Abstract
Carmine has been used in biological staining to demonstrate selectively nuclei, chromosomes or
mucins, depending on the formulation. Throughout its history in science, complaints and
frustrations have been expressed about dye quality. Inconsistencies in dye quality or identity have
prevented thorough understanding of staining mechanisms and have caused many stain solutions
to behave unsatisfactorily. The aim of this review is to (1) detail causes of these problems, which
are rooted in history, geography and production, (2) offer ways to minimize problems and (3)
provide modern explanations for stain behavior. Carmine is a ‘‘semi-synthetic’’ dye, i.e., a
complex of aluminum and the natural dye cochineal (carminic acid). Carmine shows considerable
batch-to-batch variability. Geography, politics, history, agricultural practices and iconography all
contribute to the variability of cochineal. In addition, widely divergent manufacturing methods
are used to produce carmine. Also, confusion in terminology has led to mislabeling. Pressure
from the food industry for a more satisfactory colorant for acidic foods led to the introduction of a
new dye, aminocarminic acid, which could enter the biological market inadvertantly. Improved
methods of analysis should help the certification process by the Biological Stain Commission.
Further standardization could be achieved by replacing most of the methods of solubilizing
carmine. The majority of these methods use heat, which is likely to damage the dye molecule.
Fortunately, carmine is readily dissolved by raising the pH of the aqueous solvent above 12, and a
new form of the dye, now available commercially, is soluble in water without the need for heat or
pH adjustment. Chemical structures and physical properties of carminic acid, carmine,
aminocarminic acid and kermesic acid are reviewed. A new configuration for carmine is
proposed, as well as possible changes to carminic acid and carmine molecules as a result of
decomposition caused by heating. Each of the major classes of carmine-based stains is described
as are possible mechanisms of attachment to specific substrates. Glycogen binds carmine through
hydrogen bonding, and it is here that carmine decomposed by heat could have the greatest
detrimental impact. Nuclei and chromosomes are stained via coordination bonds, perhaps
supplemented by hydrogen bonds. Finally, acidic mucins react ionically with carmine. Specificity
in the latter case may be due to unique polymeric carmine molecules that form in the presence of
aluminum chloride.

Key words: acetocarmine, aminocarminic acid, Best’s carmine, carmine, carminic acid, cochineal,
kermes, kermesic acid, mucicarmine

A full account of carmine must include coverage of
its parent compound carminic acid, which in turn
Address for correspondence: Richard W. Dapson, Ph.D., Dapson
& Dapson LLC, 6951 East AB Avenue, Richland, MI 49083.
E-mail: dick@dapsons.com
– Biological Stain Commission
Biotechnic & Histochemistry 2007, 82(4
5): 173
187.
comes from the legendary colorant cochineal.
Cochineal and its derivatives have a long history
shrouded in political intrigue and embellished by
folklore. From its earliest use by Aztec, Mayan and
Incan peoples through the subsequent Spanish
monopoly on cochineal trade, confusion has
reigned regarding origin, extraction and use. Cur-
iously, biological scientists today still cope with

DOI:10.1080/10520290701704188 173
 uncertainties regarding these substances, including
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how modern producers make them and thus what
exactly constitutes the dye called carmine.
Much of this review is based on published
scientific work, but other material has been gar-
nered from commercial and academic websites and
even from personal conversations with producers
whose anonymity is maintained to protect their
interests. In all cases, I have made every effort to
corroborate these sources and to include here
only material that I consider reliable. Books by
Garfield (2000), Findlay (2004) and Greenfield
(2005) were immensely helpful in compiling histor-
ical information.
Dye names and identity
Beyond the myriad trade names, the naming of
dyes within the cochineal family is confusing.
Historically, the term cochineal was used indiscri-
minately for any or all of them. Some producers,
extractors and suppliers today are only slightly less
ambiguous. Naming customs differ among the
food, cosmetic, drug, art supply and textile indus-
tries. This situation can lead to accidental mis-
branding by downstream vendors who bring the
dyes into unrelated fields. Intentional misbranding
also occurs either to protect trade secrets or to avoid
regulatory issues. The latter practice creates pro-
blems for biologists and chemists seeking factual
information, because virtually all carmine is made
for nonscientific use. Terms like ‘‘cochineal pro-
ducts’’ (both cochineal and carmine), ‘‘cochineal
extract’’ (carminic acid) and ‘‘acid-stable carmine’’
(either carmine or aminocarminic acid) appear in
producers’ catalogs without clarifying descriptions.
I have been able to discern useful meaning to the
names only by chemical analysis or through con-
fidential conversations.
For the purposes of this review, the names
cochineal, carminic acid, carmine and aminocarmi-
nic acid will be used in the strictest senses possible.
Cochineal is the crude material derived from the
dried bodies of female cactus insects. Carminic acid
is the active color ingredient of cochineal and may
appear occasionally as an article of commerce in
scientific markets. Carmine is a metal coordination
complex involving aluminum and carminic acid
with varying amounts and types of other com-
pounds, some of which are vital to specific uses of
the product. Carmine generally is produced di-
rectly from cochineal, not the more purified carmi-
nic acid. An insoluble pigment sold as carmine is
carmine, as just defined, precipitated with calcium
or other metal ions.
174 Biotechnic & Histochemistry 2007, 82(4
5): 173
187
Aminocarminic acid, which recently has become
available commercially, is a derivative of carminic
acid that imparts a deep red color to acidic foods
that is superior to shades possible with carmine or
carminic acid. Its existence was first revealed by
investigators in the food industry (Kawasaki et al.
2002), but most product literature does not disclose
its true identity. Because it is not widely approved
for use in food, aminocarminic acid is a well kept
secret, sometimes posing as acid-stable carmine,
which is a term with little meaning. Because
aminocarminic acid is made by reacting carminic
acid with ammonia, a similar reaction is possible
with carmine, the product of which would be the as
yet unnamed aminocarmine.
The Colour Index (CI) identifies carminic acid as
natural red 4, CI 75470. The other dyes are not
given standardized designations, although carmine
is mentioned (somewhat erroneously, see below) as
the aluminum-calcium lake of carminic acid. Che-
mical Abstracts Service registers carminic acid as
1260-17-9 and carmine as 1390-65-4. The Interna-
tional Numbering System for approved food colors
in the countries of the European Union and else-
where lists E120 for carminic acid, although I have
seen product literature in which E120 is also
applied to carmine and aminocarminic acid.
Natural history of cochineal
Raw cochineal is a blood-like fluid within the body
of the cochineal insect, a scale insect, Dactylopius
coccus (also known as Coccus cacti) of the order
Homoptera. Common names for the insect include
cochineals and cochinilla. Dactylopius is a parasite
of certain species of prickly pear cactus (Nopalea
and Opuntia) often called nopals (although nopal
literally means cactus, the word is also used in this
more restricted sense). Nopalerias are plantations
or small gardens devoted to the cultivation of these
cacti for harvesting cochineal.
Although the insects feed on nearly any of the
150 species of prickly pear, only 19 have proved
useful for commercial purposes. Opuntia indicamil
is the preferred host (Portillo and Vigueras 1998b).
Natives of Mexico and South American have
practiced artificial selection of the cacti and insects
together for centuries, producing highly productive
strains unique to specific geographic regions.
Female cochineals of the selected strains attain a
larger size and contain a purer dye than males or
wild types. These cultivated forms, however, are
vulnerable to slight shifts in climate, making them
nearly impossible to translocate to other geo-
graphic regions, a lesson learned by the British
 and French only after centuries of futile clandestine
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attempts.
Adult gravid females range from 4 to 6 mm in
size and are immobile; adult males are smaller and
winged. Fertilized females produce mobile nymphs
that exude long waxy threads that serve as para-
chutes. After an initial feeding, nymphs crawl to
the edge of the cactus pad and are dispersed by the
wind. Once established on another plant, the
nymphs mature. Females become stationary and
males develop wings and disperse again, thereby
ensuring greater genetic diversity. Nymphs and
adult females become covered with white waxy
powder that protects them from exposure to the
sun and rain.
Carminic acid apparently functions in nature to
deter predation by other insects (Eisner et al. 1980),
although some, a fly, a beetle and a caterpillar, do
feed on cochineals and appropriate the dye for
their own protection (Eisner et al. 1994). When
disturbed, these predators emit blood, rectal fluid
or crop fluid laden with carminic acid, thus
deterring their own predators. The protective
efficacy in cochineals is problematic, however, as
Portillo and Vigueras (1998b) list numerous exam-
ples of insect predators of cochineals, some of
which are known to have decimated cochineal
production in both modern and colonial times
(late 1700’s) in Mexico. A recent outbreak of needle
worm (Neuroptera: Hemerobidae: Sympherobius
sp.) in Peru destroyed up to 40% of the cochineal
crop (Portillo and Vigueras 1998a).
Geopolitical history of cochineal
Findlay (2004) and Greenfield (2005) provided
extensive and authoritative reviews of the geopoli-
tical history of cochineal. Before the discovery of
the New World by Europeans, kermes (kermesic
acid, natural Red 3, CI 75460) was the only rich red
dye known in Europe and the Middle East. It was
obtained from minute larvae found in oak galls and
scale insects found throughout most of Europe, but
the finest grade came from Aleppo, Syria. Because
of its extremely high cost, use of kermes was
restricted to the most privileged economic, govern-
mental and religious classes. All other needs for
red dye, including textiles for the masses, cosmetics
and artists’ pigments, were met by madder ex-
tracted from the roots of the madder plant (Rubia
tinctorum). Madder (alizarin, CI 58000) fails to
achieve the brilliant saturated hue characteristic of
kermes, especially when kermes was derived from
Aleppo’s oak galls. Tannins in the oak tissue are
ingested by the parasitic larvae and form a self-
mordanting dye.
When Spanish explorers discovered and Con-
quistadors invaded Central and South America in
the late 15th and 16th centuries, they found well
developed civilizations with rich natural resources,
including an unusually fine red dye. Immediately,
global trade in cochineal became enormous. Be-
tween 1575 and 1600, exports to Spain ranged from
50 to 160 metric tons annually (Findlay 2004); only
gold and silver were more important. Ironically,
taxes to Spain were paid in grana, unprocessed
dried cochineals, just as the Middle East paid Rome
during Biblical times using grana consisting of
kermes insects.
Spain went to great lengths to protect its trade
monopolies over a period of 300 years marked by
almost continual warfare with England, France and
other countries. England authorized seamen to
plunder Spanish ships, creating a loose naval force
of privateers. Most of the shipping routes from
Spanish territories in the New World passed among
the islands of the Caribbean, which served as the
bases for these privateers. Losses from Spanish
ships were substantial, although many voyages
certainly were successful and allowed Spain to
hold a huge market share in the cochineal trade.
Substantial amounts of pirated dye ended up on the
European black market, however. These events had
repercussions far from Western Europe and the
Americas. Cochineal shipped to the Middle East by
Spain rapidly replaced kermes, which doomed the
local economy of dyemakers. The dye caused a
cultural upheaval as well, because the upper classes
lost their unique red status symbol.
While Spain maintained a near monopoly on its
red dye, competing nations made numerous at-
tempts to acquire cochineal through espionage and
outright theft. Some of these stories make highly
entertaining reading (Thierry de Menonville 1787).
None were successful, however, in part because the
origin of the dye was a complete mystery for nearly
200 years, even to the Spaniards. Those who tried to
steal the secret hardly knew where or for what to
look. Most thought cochineal was derived from
various plants. Once Thierry de Menonville dis-
covered the real origin, non-Spanish Europe was
still unable to establish nopalerias. The general
strategy was to plant prickly pear cactus any-
where it could be grown (even in India), then
parasitize the plants with cochineals stolen from
some place in the Spanish New World. Because
New World plantations were heavily guarded,
thieves generally stole wild cochineals, not realiz-
ing that their dye content was insufficient for
Carmine: history, chemistry, modes of action 175
 commerce. These attempts never succeeded be-
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cause the insects either died out or produced an
inferior, worthless dye. It took many decades
before anyone realized the real secret that specific
strains of cochineals and cacti, both of which had
been deliberately optimized for local geographic
compatibility were required. Even if the optimized
strains of cochineals had been obtained with their
host cacti, their fate in other geographic regions
would have been problematic.
Modern commercial cochineal is harvested pri-
marily from plantations in Peru, Chile, Bolivia and
the Canary Islands. Small amounts also come from
southern Spain, Algiers and Australia. Mexico,
once a major center of production, suffered near
total loss of the industry when aniline dyes
appeared in the 1860’s. It currently is being re-
established beyond cottage industry status,
although progress is slow. Ironically, the factor
that led to cochineal’s demise also is the one
responsible for its worldwide resurgence. Synthetic
dyes, particularly red ones, were tied to a number
of health issues in the 1980’s, particularly in the
United States; Japan and Europe soon joined in
banning many of them from food, drugs and
cosmetics. Cochineal derivatives have become the
most popular red colorants for these purposes,
putting serious pressure on countries that can raise
high quality insects.
The symbolism of red persists to this day, serving
as an icon for authority and sometimes implying
wealth. Roman Catholic cardinals are cloaked in it.
Red neckties (‘‘power ties’’) in the business world
symbolize importance and leadership. Interest-
ingly, none of this was lost on then-President-Elect
Evo Morales of Bolivia. According to news reports
(CNN.com, January 21, 2006), he appeared at a
spiritual, pre-inaugural ceremony ‘‘dressed in a
bright red tunic worn only by the most important
pre-Inca priests.’’
Production of cochineal and its
derivatives
Variation in the quality of cochineal is a serious
problem for scientific users. Differences in climate,
soil type, moisture, season and geography affect
the quality and composition of the cochineal to the
extent that geographic origin sometimes can be
determined by spectrophotometric, chromato-
graphic and statistical tools (Mendez et al. 2004).
Processors who restrict their starting material to a
prescribed geographic area have less variable dyes
than those who buy dried insects on the open
market. There are three distinct supply chains, each
176 Biotechnic & Histochemistry 2007, 82(4
5): 173
187
having a significant impact on the quality of
cochineal.
1. In the traditional method, independent
growers sell dried cochineals on the open
international market. Buyers of the raw mate-
rial store it in central warehouses, often in
another country, and eventually sell it to
companies that process it into cochineal extract
or carmine. This is the model used historically
by the Spanish monopoly for nearly 400 years,
and it continues to be the most common
practice today. Peru is the largest cochineal
grower, exporting more than half its product
as dried insect bodies. The problem with this
supply chain is that neither the processor nor
the end user has control of the quality of the
dye or batch-to-batch variability. This distribu-
tion method has had negative economic con-
sequences for the growers, which further
compromises product uniformity. Because
few of the rich profits are returned to them,
they remain in poverty, and phase in and out of
production.
2. The second supply chain developed from an
attempt to consolidate profits more at the local
level. Various government foreign aid pro-
grams have supported establishment of pro-
cessing plants near independent cactus
plantations. The local economy benefits sig-
nificantly and there is a more reliable supply of
dye, because there is a reliable local buyer. An
unintended consequence, for scientists at least,
is that geographic variability is reduced. Boli-
via is leading the way with this model (Inter-
national Development Research Centre 2006).
3. At least one company in Chile has chosen to
consolidate the supply chain under a single
corporate entity. Agricultural production, ex-
traction, purification, synthesis and finishing
as a powder or liquid are all conducted at a
single site by a dedicated work force. This
provides the best possibility for uniform qual-
ity and consistent supply.
Actual production of dye begins with the har-
vest. Ideally, only gravid cochineals are brushed off
cactus pads, leaving most of the immature forms
and spent females behind. This is not a simple task,
because the females and prior offspring are densely
intermingled within a waxy white exudate. Indis-
criminant harvesting of the masses is faster than
careful selection of gravid females, but produces an
inferior product with lower dye content and more
 impurities. Premium cochineal contains only
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gravid females.
Methods of killing and drying the cochineals
vary widely and are said to affect the dye content,
although to my knowledge this has not been
verified. Insects may be killed in boiling water or
by drying in the sun or in ovens. Modern opera-
tions use carefully controlled heating methods to
ensure uniform quality, although oven-killed and
sun-dried material is still popular in less developed
areas. Much lore and proprietary obfuscation is
involved in many of the cochineal stories, ancient
and modern. We simply cannot know for certain
what happens to the raw cochineal from this point
on. Microscopically, high quality cochineal appears
to contain body parts of the dye-rich females, but
little else (personal observation). Hastily harvested
material also contains insect secretions, plant parts
and body parts of dye-poor individuals. These
contaminants affect the yield of carmine in sub-
sequent processing, because most of them are
insoluble. Dye purity is affected as well if soluble
contaminants are present in significant quantity.
The fineness of the grinding affects efficiency of
extraction.
The extraction solvent may be water or an
aqueous ethanol solution, either of which is made
alkaline with sodium or potassium hydroxide or
carbonate, or ammonium hydroxide. At least one
process, apparently borrowed from ancient pro-
duction methods for kermes, uses water acidified
with acetic acid. The extraction liquor may be dried
or sold as a liquid, typically under the names
cochineal or cochineal extract, respectively. It con-
tains carminic acid, soluble proteins, carbohydrates
and other biological molecules from the insects, as
well as various ionic salts from the extraction
process. In Spain, the waste stream from some
processors contains high levels of phosphate and
ammonium (Chimenos et al. 2003), leading one to
suspect that phosphate salts may be present in the
product and that amination might have occurred
(see below). I have been unable to find any
information that suggests the use of phosphate in
the production of South American carmine.
Carminic acid may be purified further from the
cochineal extract and sold to the scientific commu-
nity, but it is expensive and rarely used to make
biological stains. Carmine is derived from the
initial cochineal extract, not purified carminic
acid, using alumina hydrate (aluminum hydroxide)
to form an insoluble aluminum lake. This product
may be processed further to yield a powder that is
freely soluble in water.
Calcium ions may be used to assist precipitation
of the lake, but some processors provide a calcium-
free product. Until recently, most of the structural
formulas for carmine included calcium (e.g., Green
1990, Meloan et al. 1971); however, when it is
present, calcium probably is simply a component
of the mixture sold as carmine and not part of the
molecular structure of carmine (see below).
Consequences of variability
Clearly, carmine is one of the more variable dyes on
the biological stain market. Causes of this varia-
bility, are discussed above and summarized in
Table 1. It is easy to understand why this dye has
frustrated both researchers and clinical users.
Meloan et al. (1971) noted that ‘‘Investigations of
carmine were rendered difficult by wide variations
in the staining properties of dye samples and the
lack of data concerning the composition of various
batches of carmine.’’ Thirty-five years later, we
continue to hear histotechnologists complain that
their carmine-based stains, both commercially pre-
pared and lab-made, are too weak or have un-
expectedly short shelf-lives. Perhaps related to this
is the comment that they cannot dissolve their dyes.
One dye lot works beautifully, another is unsatis-
factory. Recognizing these frustrations, Horobin
and Bancroft (1998) advised finding a good carmine
lot, then stocking up on it.
In other cases, the same lot of dye seems to
perform well for some people, but not for others.
This suggests that certain stain preparation techni-
ques may be harmful to the dye. Different methods
of dissolving the dye may have a significant impact
on dye content and performance of the solution,
Table 1. Summary of the sources of variability in the
quality of carmine-based stain solutions.
Cochineal
Geography
Environment
Season of collection
Collection techniques
Killing and drying techniques
Grinding techniques
Carmine and carminic acid
Supply chain (single- vs. multi-sourcing)
Extraction techniques
Purification techniques
Derivatization techniques
Carmine stains
Solubilizing techniques
Stain formulas
Carmine: history, chemistry, modes of action 177
 either by being more or less effective, by degrading
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the dye through overheating, or by aminating it by
the use of ammonium hydroxide. Further con-
founding the issue of dissolvinig the dye is its
variable ionic state. A method for dissolving the
dye that works well for one sample may be a poor
choice for another. This set of problems is easily
avoided. Unless the dye is the barium salt, raising
the pH to 12.5 with sodium hydroxide converts all
forms to the highly soluble sodium salt. Excessive
heating or boiling of solutions then is not necessary.
I favor using warm (B 608 C) deionized water.
Standardizing this single step in stain preparation
would eliminate most of the problems unrelated to
dye purity. Using the water soluble sodium salt of
carmine (EZ Carmine; Anatech Ltd., Battle Creek,
MI) simplifies the procedure even more, because
there is no need to adjust pH to get the dye into
solution.
Identity and purity are certainly problematic
with carmine. Mislabeled dyes are a problem
with uncertified material, but that is avoided easily
by using only dye certified by the Biological Stain
Commission. There is a possibility that impurities
in the dye could affect the performance of a stain
solution. Because carmine-based solutions stain
tissue elements by various physico-chemical means
(see below), a soluble impurity may interfere with
or enhance one type of stain and not another.
Certification of carmine by the
Biological Stain Commission
Because of the uncertainty surrounding the con-
stitution of carmine and the difficulty in obtaining
stable solutions with consistent hue, no assay
procedures were included in the certification pro-
cess of carmine by the Biological Stain Commission
until 2007. A spectrophotometric analysis (Dapson
et al. 2007) now is performed in conjunction with
tissue testing using Orth’s lithium carmine nuclear
stain, Southgate’s mucicarmine and Best’s stain for
glycogen (Horobin and Kiernan 2002).
Analytical procedures
Proper analysis of a sample should provide an-
swers to two questions: what dye is contained in
the sample, and how much of it is there? Marshall
and Horobin (1974) detailed methods designed to
identify carminic acid and carmine by thin layer
and gel filtration chromatography. They also quan-
tified these dyes in various samples. Carmine was
first dissolved in ammonium hydroxide. Both dyes
then were boiled under reflux for 10 min using 0.02
178 Biotechnic & Histochemistry 2007, 82(4
5): 173
187
M hydrochloric acid. Absorbance was measured at
490 nm. In light of more recent knowledge that the
sugar moiety is labile at high temperature (Green
1990, Lancaster and Lawrence 1996) and that
ammonium hydroxide might aminate the molecule
(Sugimoto et al. 2002), these procedures are ques-
tionable. With the potential for encountering ami-
nocarminic acid, there is even greater impetus for
new analytical methods.
Spectrophotometry has been tried by others with
little success, but apparently this is because re-
searchers failed to appreciate how color changes
over a broad range of pH. For all of these dyes, any
pH between 3 and 11 is unstable, because small
changes in the acid content of the solution effect
large shifts in pH. Not surprisingly, it is in this
range also that color changes are most pronounced,
resulting in dramatically shifting absorbance
curves (Fig. 5).
The new assay can identify the three dyes and
provide a relative quantitative assessment of purity
(Dapson 2006). The sample is dissolved volumetri-
cally in water to which enough 1.0 N sodium
hydroxide is added to raise the pH to 12.5
12.6.
This converts the dye to the sodium salt, if it was
not in that form already, and brings the pH to a
stable plateau on the dissociation curve. Dominant
peaks are recorded from a spectrophotometric scan
in the visible range. An aliquot of the sample then
is treated with 20% hydrochloric acid to lower the
pH to 1.9
2.1 (another stable plateau), and the scan
is repeated. Carmine has a dominant peak at
530
535 nm at high pH. The other two dyes have
closely similar dominant peaks at high pH, viz.,
565
570 and 570
575 nm for carminic acid and
aminocarminic acid, repectively. They are distin-
guished readily at low pH, however. The peak for
carminic acid is 490
500 nm, while that of amino-
carminic acid is 525
530. This procedure clearly
identifies the dyes. Peak absorbance at high pH
provides a relative measure of dye content.
Assays of samples not certified by the Biological
Stain Commission revealed a troublesome degree
of mislabeling; carminic acid was sold as carmine
and vice versa. Nearly every lot certified by the
Biological Stain Commission (N  81) was assayed;
all proved to be carmine. There was significant
variation in dye content among the certified sam-
ples, however. Spectrophotometric absorbance ran-
ged from 1.11 to 1.87 with a mean of 1.36.
Absorbance has been shown to be related linearly
to concentration under the conditions of the assay,
thus we can relate these numbers to dye content in
a relative way to gain a better appreciation of
the magnitude of the variation. If someone had
 purchased the high content lot to make stain
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solutions, then obtained the low content lot later,

the new stain would have had just over half (60%)
of the dye content of former solutions.
Samples from the Biological Stain Commission
had been certified over a period of nine decades.
Since the 1960’s, dye purity has decreased signifi-
cantly. Samples from the last two decades con-
tained eight of the 10 lots with the lowest dye
content since 1920 (Dapson 2006).

Stability

The stability of dry carmine dye over time appar-

ently has never been investigated. Judging from the
satisfactory results of staining with 40-year-old
samples in my lab, the dye seems to store well.
Now that most samples of carmine certified by the
Biological Stain Commission have been assayed,
they should be re-analyzed some years in the
future to verify my observation. Certainly, lots
from recent decades that showed such high varia-
bility warrant closer scrutiny.

Chemical structures and physical

properties
Cochineal
Unprocessed cochineal contains the active ingredi-
ent carminic acid (see below) and other biological
molecules as well as body parts, waxy exudate and
residue from the environment. While most of the
extraneous matter is insoluble, aqueous solutions
(cochineal extract) contain some undenatured pro-
teins and carbohydrates. Cochineal is available
commercially in a variety of forms: unprocessed
(insect bodies), coarsely crushed, finely powdered,
crystallized, or liquified. None of the solid forms is
soluble in water to any significant extent, but can
be readily dissolved by adjusting the pH above
11 or below 3. Any of these solutions is stable,
although color varies with pH.
Carminic acid
Carminic acid has an anthraquinone nucleus with
carboxylic acid, methyl group, four hydroxyls
and hexose sugar moieties attached. The currently
accepted structure (Fig. 1) is based on the work
of Bhatia and Venkataraman (1965). The molecular
formula is C22H19O
13 and the molecular weight
is 491, both of which are for the ionic species.
The three-dimensional conformation, rendered
by molecular modeling software after energy
Fig. 1. Structure of carminic acid. The hexose moiety is
shown in red. Color scheme for three-dimensional model:
gray, carbon; black, hydrogen; green, oxygen.
minimization (Dapson 2005), is a slightly curved
plane (the anthraquinone nucleus) to which is
attached a bulky globular mass, the sugar moiety.
Most of the molecule is studded with oxygen atoms
and hydroxyl groups, creating ample opportunity
for hydrogen bonding.
Because of the positioning of the carbonyl and
hydroxyl groups, carminic acid is ideally suited for
coordination bonding with metals, thus creating
carmine. Indeed, nearly all historical and current
uses of carminic acid for textile and biological
purposes involve complexing with aluminum or,
more rarely, iron or tin. Insoluble pigments for
printing and art usually are barium salts of
carminic acid.
Carminic acid available to the scientific commu-
nity during the last 60 years has been either the free
acid or the sodium/potassium salt. Of seven
samples from an archival collection available to
me, four were soluble in water at any pH and must
have been the salt. The remaining three were
insoluble in water unless the pH was adjusted
above 11, indicating that they were in the free acid
form. Only one sample was labeled ‘‘carminic acid,
sodium,’’ the remainder gave no indication of their

Carmine: history, chemistry, modes of action 179

 ionic nature. Two of the samples were labeled
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erroneously as ‘‘carmine’’ (Dapson 2006).
Carmine
Molecular formulas for carminic acid have been in
dispute for well over a century, so it is not
surprising that the structure of carmine has been
problematic (reviewed by Meloan et al. 1971). The
most likely structure for carmine contains two
molecules of carminic acid coordinately bound to
a single aluminum atom at carbonyl-hydroxyl pairs
as in Fig. 2. The structure in Fig. 2a was proposed
by Meloan et al. (1971), based on comparable
structures of alizarin (Kiel and Heertjes 1963a,b).
Alternative configurations using one or two alumi-
num atoms with or without calcium were sterically
impossible with Stuart-Briegleb atomic models.
There are two problems with the conformation
proposed by Meloan et al. (1971, p. 92), however. In
their atomic models, both carminic acid moieties lie
in the same plane with hexose units positioned at
the same end. Both features are implausible and
cannot be duplicated with molecular modeling
software. Bond angles for aluminum do not permit
a single plane configuration; the carminic acid
groups must lie crosswise to one another at an
angle of about 1208 (Fig. 2c). Drawn flat (Fig. 2b),
the two carminic acid groups are in the trans, not
cis, position.
Calcium has been thought to help bind the two
carminic acid groups together, but there is no
evidence for this. Certain production processes
Fig. 2. Structure of carmine. A) Two-dimensional rendition from the three-dimensional atomic model of Meloan et al.
(1971) that had both carminic acid moieties in the same plane as hexose units (red) on the same ends. B) Structure as
verified by molecular modeling software in which the hexose units are on opposite ends. C) Three-dimensional
conformation created by molecular modeling software in which the carminic acid moieties are positioned about 1208 to one
another. The large magenta atom is aluminum.
180 Biotechnic & Histochemistry 2007, 82(4
5): 173
187
(some ancient) actually avoid calcium and even
use purified water, so evidently it is not essen-
tial (Meloan et al. 1971, Muspratt 1860, Napier
1875). Calcium sometimes is used in modern
processing to precipitate carmine from solution,
in which case it would serve as the counterion in
the insoluble calcium-carmine salt. Another likely
configuration would be as a chelated species in
which calcium is coordinately bound to one or
more of the carbonyl/hydroxyl pairs .
The carmine of commerce may be the free acid or
a salt of barium, calcium, potassium or sodium.
The free acid and calcium salt are insoluble except
at high pH (]11). Sodium and potassium salts are
freely soluble in water at room temperature; the
barium salt is insoluble. Only two of eight archived
commercial samples of carmine were freely solu-
ble; one was only partially soluble, suggesting a
mixture of ionic forms (Dapson 2006). One sample
labeled ‘‘carmine’’ was actually carminic acid, and
another was aminocarminic acid.
Solubility characteristics and degrees of purity
are tailored by producers for specific markets.
Commercial carmine solutions usually are alkali-
nized with sodium, potassium or ammonium
hydroxide. Barium salts are preferred for artists’
pigments. Carmine for cosmetics typically is a
pigment also, while food and drug uses generally
require more readily soluble dye. F D & C carmine
for food, drug and cosmetic use must contain at
least 50% carminic acid. Because carmine used for
the biological stains market can be from any of
these sources, it is no wonder that carmine has had
 a reputation in the biological community for lack of
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uniformity.
Carmine solutions are stable in light and resist
oxidation. At low pH (B 3), carmine precipitates
slowly as the free acid, so it cannot be used in
acidic foods. In those applications, carminic acid
(cochineal extract) or aminocarminic acid must be
used.
Aminocarminic acid
Although carminic acid and cochineal extract can
be used at low pH, they lose their rich red hue,
turning reddish orange or even orange. Acid-stable
derivatives now are widely available to the food
industry in liquid and solid form, but their identity
and methods of synthesis are tightly guarded
secrets. One likely molecular structure is 4-amino-
carminic acid (Fig. 3), purportedly made by expos-
ing carminic acid to hot ammonia solution
(Kawasaki et al. 2002, Sugimoto et al. 2002). More
extensive amination certainly is possible in a
manner reminiscent of the syntheses of amino-
orceins by Beecken et al. (2003). Questions abound
concerning the extent of amination, however. Are
all such aminated products of commerce made
Fig. 3. Structure of aminocarminic acid as determined by
Sugimoto et al. (2002). The amino group (blue) has
hydrogen bonding capability comparable to the hydroxyl
and carbonyl groups. More fully aminated species are
likely.
from carminic acid or is it possible that some really
are aminated carmine? Can aminocarminic acid be
made into aminocarmine in a process like that used
to convert carminic acid to carmine? The commer-
cial names aminocarmine and acid-stable carmine
suggest that the dye is not simply aminocarminic
acid. In the rare instances when the trade literature
deals with the chemistry of the substance, however,
the term aminocarminic acid is used. All three
samples that I assayed had similar spectrophoto-
metric characteristics, although their labels read
‘‘acid-stable carmine’’ or ‘‘carmine’’ (Dapson 2006).
All were intended for use in food.
Because this dye is used widely by the food
industry, it is possibility that it already has entered,
or eventually will become available to, the biologi-
cal stain market. Most manufacturers and suppliers
do not reveal its true composition, simply labeling
it acid-stable carmine or some trade name that
suggests its stability in acid environments. Labels
and product literature give it the same official
designations as carmine (natural red 4, CI 75470,
EEC Code E120) despite structural differences. This
possibly is a ruse to get around regulations directed
at colorants in food; at least one country, Japan,
already has banned its use (Sugimoto et al. 2002).
An important question is whether this dye is
suitable for use in conventional biological stains
calling for carmine. Further research is needed to
establish the suitability of aminocarminic acid in
histological formulations. It can be identified read-
ily by spectrophotometry (see below).
It should be noted that ammonium hydroxide
ocassionally is used for initial extraction or solubi-
lization of cochineal. This process may produce an
aminated molecule. If so, aminocarminic acid of
some type may have been with us all along. This
same consideration arises again when we make
Best’s carmine with ammonium hydroxide or
perhaps even Mayer’s carmalum with ammonium
alum. Do these stains contain carmine or amino-
carmine?
Kermesic acid
Kermesic acid (CI 75460, CI natural red 3) is the
active colorant in kermes, the natural red dye
produced by several insect species in Europe and
the Middle East. It is similar to carminic acid except
that it lacks the carbohydrate moiety (Fig. 4) and it
seems to have been used in similar ways
for hundreds of years. This suggests that the
carbohydrate portion may be superfluous for either
color or function, at least in some applications.
Interestingly, Green (1990) cautions that heating
Carmine: history, chemistry, modes of action 181

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Fig. 4. Structure of kermesic acid, the active colorant of
kermes. Lacking the hexose moiety of carminic acid, this
dye has fewer potential sites for hydrogen bonding.
carminic acid may eliminate the carbohydrate from
the anthraquinone nucleus, and Lancaster and
Lawrence (1996) confirmed that such decomposi-
tion occurs above 1008 C. Because historical meth-
ods for commercial dyeing involved prolonged
boiling (Bemiss 1815), nearly all current industrial
processes use heat, and most directions for making
histological stains call for boiling, or at least
heating, one must wonder if carmine contains
carminic acid, kermesic acid or a mixture of both.
Perhaps it makes no difference to either textile
dyeing or biological staining, but see below.
Mechanisms of staining
Four distinct types of solutions dominate the
literature for carmine-based stains for plant and
animal histology: Best’s stain for glycogen, Orth’s
lithium carmine and carmalum solutions for nuclei,
aceto-carmine for chromosomes, and mucicarmine
for certain mucins. Obviously, the varied substrates
for these stains are chemically distinct, so how can
one dye be used to stain each so selectively? While
little is published about how the stains work, we
can readily surmise that different conditions un-
ique to each type of stain (pH, ions etc.) permit
different chemical mechanisms to operate. This
section deals with how various ingredients influ-
ence divergent reaction mechanisms.
182 Biotechnic & Histochemistry 2007, 82(4
5): 173
187
The first step is to understand what is and what
is not a probable staining mechanism. Bonding
parameters derived from quantum mechanical
calculations by molecular modeling software pro-
vide that information. There are seven ways for
dyes to bond to tissues: covalent bonding, ionic
bonding, coordinate bonding involving metal
atoms, hydrogen bonding, and three types of
van der Waal’s forces (Keesom, Debye and London
attractions), explained in some detail by Dapson
(2005). Bonding parameters measure the potential
to engage in most of these mechanisms (covalent
bonding is not included and coordinate bonding is
indicated by the presence of metal atoms).
Carmine contains at least one aluminum atom
capable of engaging in coordinate bonding. The
dye has no potential for covalent bonding to tissue
sites. Because the structure of carmine may not
be the same in different stain formulations, the
bonding parameters for carminic acid will be used
instead to assess other bonding mechanisms.
Table 2 lists these parameters and compares them
with more than 400 other dyes used for histology
(Dapson 2005).
Glycogen
Best’s carmine stain was long considered to be
empirical (Sheehan and Hrapchak 1980) despite
convincing evidence that hydrogen bonding was
the most likely staining mechanism (Horobin and
Murgatroyd 1971). With the highest hydrogen
bonding parameter of any dye studied (Table 2),
carminic acid, hence carmine, certainly has the
potential for engaging in hydrogen bonding. In its
ionic form, carmine has 18 potential sites for
hydrogen bonding, assuming that one of the
structures depicted in Fig. 2 is correct. Likewise,
glycogen is richly studded with similar sites.
Unquestionably, the potential is there, but are
reaction conditions favorable for selective staining?
Best’s formula (Sheehan and Hrapchak 1980) has
a high ionic content (potassium carbonate and
chloride) and about 38% methanol. These factors
tend to suppress ionic bonding via the cationic
aluminum atom, thus any background or nonspe-
cific staining would be unlikely. Glycogen has
comparable potential for hydrogen bonding. The
‘‘differentiating solution’’ (36% ethanol, 18%
methanol, 45% water) immediately following the
stain is safe for extracting unbound dye, although
its high water contact removes bound dye over
time. Directions caution against following the stain
with water (Horobin and Bancroft 1998), which
 Table 2. Bonding parameters for carminic acid. High values indicate the potential for engaging in a particular type of
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bonding. For comparative purposes, ranges are given for over 400 dyes used in histology (Dapson 2005).

Parameter Bonding mechanism Carminic acid Histological dyes

Z (ionic charge) Ionic bonding 1 ev 6 to4 ev
h (hydrogen bonding groups) Hydrogen bonding 11 0 to 11
d (dipole moment) Keesom & Debye forces 22.1 D 0 to 108.4 D
a (polarizability) Debye & London forces 266 69 to 961
X (halogen atoms) London forces 0 1 to 8
HI (hydrophobic index) Debye & London forces 5.0 1.3 to 9.4

would outcompete the dye for hydrogen bonding
sites on the substrate.
Most authorities recommend the periodic acid-
Schiff (PAS) reaction over Best’s carmine for glyco-
gen. The reasons given most often are that
the former is based in histochemical certainty while
the latter is ‘‘empirical.’’ More convincing is the
argument that PAS demonstrates more glycogen
more reliably. The basic problem with Best’s stain is
the uncertain purity and content of the carmine dye.
It is worth noting here the possible effects on
staining if the carminic acid portions of carmine
decompose to kermesic acid. Each carminic acid
originally has 11 hydrogen bonding sites (all of the
hydroxyls and carbonyls; Fig. 1). When combined
with aluminum to form carmine in a 2:1 coordina-
tion complex, each carminic acid moiety loses two
sites, so carmine has 18 positions for hydrogen
bonding (Fig. 2). If heating the dye causes loss of
both sugar groups with their combined six sites,
the ‘‘kermesic carmine’’ is left with only 12 hydro-
gen bonding sites (Fig. 6). That is still a large
number compared to other dyes; however, it is a
very different dye than unmodified carmine when
it comes to hydrogen bonding, especially if steric
factors are considered. It is not enough simply to
have potential bonding sites on a dye; they must be
placed to complement the position of reactive sites
on the tissue substrate. Glycogen is composed of
sugar molecules. Carmine itself has two sugar
groups appended to it that match the size, shape
and spacing of bonding sites on the tissue. Without
the hexoses, decomposed (‘‘kermesic’’) carmine
might exhibit weaker staining than normal car-
mine.
Nuclei
There are several formulas for carmine-based
nuclear stains (Lillie and Fullmer 1976). Most of
them probably bond to tissue sites via the alumi-
num ion within the carmine molecule. Orth’s
lithium carmine, containing only carmine, lithium
carbonate and water, is a high-pH solution that acts
as a regressive stain. Both cytoplasm and nuclei
stain, but differentiation is achieved in 1% hydro-
chloric acid in 70% alcohol. Cytoplasmic carboxyl
groups become undissociated in this acid and

Fig. 5. Shifts in color with changes in pH. For all three dyes, shades and intensities of colors generally vary within each
zone until the pH drops below 3. Carmine slowly precipitates below pH 3.

Carmine: history, chemistry, modes of action 183


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Fig. 6. Hypothetical structure of ‘‘kermesic carmine,’’
carmine whose hexose moieties have been lost.
destain, while the phosphoric acids of DNA and
RNA remain ionized and retain the stain. The
question is just what is being stained. If carmine
retains its aluminum atom in a positively charged
state, bonding would be to anionic nucleic acids;
however, the carminic acid moieties would be
negatively charged at high pH and could bind to
histones.
Grenacher’s formula uses ammonium alum;
Mayer’s weaker carmalum is similar, with ammo-
nium or potassium alum. Here the acidity of the
alum creates a progressive stain that prevents
carboxyl groups from staining. Obviously, either
of these is similar to several varieties of hematox-
ylin stains. Mayer also created an alcoholic version
using aluminum chloride, calcium chloride and
70% alcohol (Davenport 1964). In this case, the high
ionic content (4% calcium chloride), the alcohol,
and the low pH owing to the aluminum chloride
discourage staining of carboxylic acids. One might
argue that some hydrogen bonding to the sugar
moieties of nucleic acids is possible given the
inhibiting effect on ionic (or coordinate) bonding
afforded by the ionic strength and alcohol content
of the solution.
There is even a chrome alum carmine (Fyg’s
recipe; Lillie and Fullmer 1976) that behaves iden-
tically to chrome alum hematoxylin. Presumably,
chromium replaces aluminum in the carmine
molecule. The chromium ion then forms a more
tightly bound coordination complex with the tis-
sue, so it can be followed by acidic reagents like
Van Gieson’s elastin stain. Incidentally, chrome
alum carmine stains nuclei blue.
184 Biotechnic & Histochemistry 2007, 82(4
5): 173
187
Chromosomes
The original formulations contained only dye,
water and acetic or propionic acid, and specificity
apparently was achieved serendipidously from use
of iron dissecting needles used to tease apart the
cells. Later versions called for the addition of iron
compounds, ferric chloride being the most popular
(Davenport 1964). With about 50% glacial acetic or
propionic acid, the pH is low enough to prevent
cytoplasmic staining and the iron probably forms a
firm coordination complex with nucleic acids as it
does in iron hematoxylin stains. Like chrome alum
carmine for nuclei, the ferric ion probably replaces
the aluminum in the carmine molecule. Chromo-
somes are stained dark red with a bluish tinge
(Humason 1979).
Mucus
Mucicarmine is the most widely used carmine-
based stain in pathology today, although it is
inferior to other methods for demonstrating mu-
cins. In Mayer’s original method, the dye was
dissolved by fusing with aluminum chloride over a
flame (Sheehan and Hrapchak 1980). The resulting
black tarry mass is difficult to produce consistently,
and one wonders how much dye is destroyed
during this process. Southgate’s more rational
method solubilizes the dye by raising the pH
with aluminum hydroxide, then acidifying the
solution with aluminum chloride. The solvent is
50% ethanol. Curiously, he prescribed boiling the
solution for 2.5
3 min, although this is unnecessary
and possibly damaging. For either variant, some
workers call for diluting the stock solution with
water (tap or distilled, according to author) or with
aqueous alcoholic solutions; others use it undi-
luted. Commercial solutions usually are ready to
use. Such variations probably arose to accommo-
date differences in dye quality or the effects of
certain drastic solubilization technics..
Textbooks tend to be vague about bonding
mechanisms, saying only that mucicarmine stains
‘‘mucins’’ (Carson 1990, Sheehan and Hrapchak
1980, Totty 2002). Given what is known about the
mechanism of staining, there is little reason for
ambiguity. Mucicarmine mimics alcian blue in its
reaction with mucins (Laurén and Sorvari 1969);
thus it stains carboxylated and sulfonated varieties
of epithelial and connective tissue mucins, includ-
ing cartilage, but not neutral mucins. The mode of
attachment is through carmine’s cationic alumi-
num atom. Lillie and Fullmer (1976) state that
Cowdry was able to stain ‘‘gastric mucus’’ red with
 an undiluted stain, but no citation or further details
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were given. Because gastric mucus is neutral in
most species, this statement is suspect. I have not
been able to stain mucus in the mucous cap cells of
the stomach with mucicarmine.
Why mucicarmine is excluded from nuclei is a
far greater mystery than why it stains acidic
mucins. The carmine molecule is very large and
may be excluded from dense nuclear materials
while readily penetrating more permeable mucins
(Horobin and Brancroft 1998). On the surface,
this is reasonable, but it leads to a deeper enigma:
why do certain other carmine-based solutions stain
nuclei so well?
If we assume that all of the carmine-based stains
contain carmine as a 2:1 carminic acid: aluminum
complex as shown by Meloan et al. (1971) and in
Fig. 2, then there is no obvious reason why
mucicarmine should behave differently from
lithium or alum carmine. If carmine is unable to
penetrate nuclei from a mucicarmine solution, it
must also be excluded when used in the other
formulations. Obviously that is not the case. One
might argue that the excess aluminum in mucicar-
mine blocks the dye from nucleic acids, but
analysis of the ratios of aluminum (from added
salts) to carmine disproves that. Mayer’s original
mucicarmine has a ratio of 0.1:1 and Grenacher’s
nuclear stains have ratios of 0.1:1 and 0.15:1. Much
higher ratios are found in Southgate’s mucicarmine
(0.45:1), but either of Mayer’s carmalum solutions
have even more aluminum (0.57:1 and 0.6:1).
Furthermore, aluminum tends to supress staining
of mucin, not nuclei, at least in hematoxylin
solutions. Harris hematoxylin, which does not stain
mucus, has more aluminum than Gill formulations,
which do stain mucus.
These varied carmine solutions differ in one
respect: the nature of the aluminum added to
carmine to make the stain. Nuclear stains either
lack added aluminum (Orth’s) or use aluminum
ammonium sulfate (Grenacher’s carmalum and
one of Mayer’s carmalums), aluminum potassium
sulfate (another of Mayer’s carmalums) or simply
aluminum sulfate (Lillie and Fulmer 1976). While
Mayer’s paracarmine uses aluminum chloride, the
solvent is 70% alcohol.
The key difference with mucicarmine seems to be
that it contains aluminum chloride. It is well
known in the wastewater treatment industry that
aluminum chloride behaves differently than the
various alums when forming polymeric complexes
Fig. 7. Proposed struc-
ture of a dimer of carmine
that might be a constituent
of mucicarmine. Larger
polymers are possible,
but if they become too
large, they precipitate.
Carmine: history, chemistry, modes of action 185
 with organic molecules in an aqueous environment.
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The process is used to precipitate organic com-
pounds from the waste stream, and of all the
aluminum salts used, aluminum chloride is the
superior compound for this purpose.
Harms (1957) proposed that by heating carmine
with aluminum chloride during preparation of
mucicarmine, additional chelates were formed at
the carboxylic acid and adjacent hydroxyl, as well
as at the carbonyl and adjacent hydroxyl. He
presented one such structure with four additional
aluminum atoms. His formula for carminic acid,
incorporated into his mucicarmine structure, is
now known to be incorrect, and he did not make
use of the new chelates to polymerize the molecule
further; however, he did recognize the potential for
new compounds stemming from the use of alumi-
num chloride. I suggest taking this concept a
logical step further: let the newly formed alumi-
num chelates of carmine polymerize (Fig. 7).
Perhaps it is the dimers and smaller oligomers
that serve as the active component in mucicarmine,
while larger polymers precipitate just as in waste-
water treatment. One only has to filter various
carmine-based stains to see that this probably
occurs. With mucicarmine, a heavy sludge coats
the paper and makes gravity filtration very slow.
The other stains filter readily and leave little
residue on the paper. With this information, Hor-
obin and Bancroft’s (1998) theory makes sense. In
carmine-based nuclear stains, the 2:1 complex is
small enough to penetrate dense nuclear material
and stains by ionic, coordinate or hydrogen bond-
ing, most likely a combination of all three. Dye
molecules in mucicarmine are at least twice as large
and are blocked from dense nuclear material. The
molecular lattice of mucus is sufficiently porous to
allow penetration and staining.
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